Abstract
Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.
Introduction
Phytoestrogens (PEs) are nonsteroidal, organic compounds that are bound by one or both of the estrogen receptor (ER) isoforms, ERα and ERβ (Kuiper et al. 1998). Although nonsteroidal, the structure of these plant-derived compounds generally includes a phenolic ring resembling the aromatic ring of ovarian-derived estrogens, such as 17β-estradiol (E2).
Endogenous estrogens affect a diverse array of tissues including the gonadotrope cells of the pituitary. These cells synthesize the gonadotropic hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which have important roles in reproduction. Specifically, in females FSH stimulates follicular growth and maturation, whereas LH promotes steroidogenesis and ovulation. Although estrogens can affect gonadotropin synthesis and secretion by influencing gonadotropin-releasing hormone (GnRH) release from hypothalamic loci, direct action of estrogens on gonadotrope cells is suggested by experiments using in vivo and in vitro models in which pituitary tissue is physically separated from hypothalamic inputs (Clarke & Cummins 1984). Indeed, effects of estrogens on basal and GnRH-stimulated gonadotropin secretion are evident in cell culture using ovine or rodent pituitary cells (Huang & Miller 1980). Although the effect of endogenous estrogens on gonadotrope function is well recognized, much less is known regarding the effect of PEs on the synthesis and secretion of FSH. This may be a particularly important concern in animal production systems, because several common forages, including alfalfa, clover, lupin, and some grains, contain high concentrations of PEs (Dixon 2004). In addition, the rumen and intestinal microbiota of domestic livestock species may enhance the estrogenic potency of feedstuffs by transforming plant precursors into active PEs (Zhou et al. 2009).
The potential effects of PEs on the reproductive efficiency of livestock is suggested by reports that female sheep and cattle grazing on subterranean clover (Trifolium subterran) or alfalfa (Medicago sativa) became infertile (Bennetts et al. 1946, Moule et al. 1963). These effects of PEs on reproduction are likely to reflect PE-dependent change in the ER function. Indeed, Mathieson & Kutts (1980) report that genistein (GN) and coumestrol (CM) compete with E2 for binding sites in hypothalamic and pituitary tissue of sheep.
In the studies reported here, we examine the physiological effect of PEs commonly present in feedstuffs provided to domestic animals. Our particular objective was to determine the effect of PEs on basal secretion of FSH. In addition, we assessed the effect of PEs on GnRH-induced LH secretion from ovine pituitary cells.
Materials and methods
Reagents and supplies
Calcium and magnesium-free (CMF) Hanks balanced salt solution (HBSS), and medium 199 (without phenol red, l-glutamine, or NaHCO3; M199) were obtained from Sigma–Aldrich. Bovine insulin, the GnRH agonist (d-Ala6, des-Gly10 GnRH ethylamide; GnRH-A), pancreatin, the ER antagonist (ICI 780 182; fulvestrant), and most PEs were also purchased from Sigma–Aldrich. The PE, daidzein (DZ), was purchased from Tocris Bioscience (Ellisville, MO, USA). The antifungal drug, fungizone, and the two antibiotics, gentamycin sulfate and penicillin/streptomycin, were obtained from HyClone (Logan, UT, USA), Mediatech (Manassas, VA, USA), and Gibco respectively. Collagenase (type-I; Worthington Biochemical Corporation, Lakewood, NJ, USA), l-glutamine (Invitrogen), BSA (fraction V; EMD Chemicals, Gibbstown, NJ, USA), and E2 (United States Biochemical Corp., Cleveland, OH, USA) were obtained from established vendors.
Cell culture
Pituitary tissue was collected from castrated male yearling sheep euthanized at a local abattoir by electrical stunning and exsanguination. Tissue was collected within 10 min of euthanasia and immersed in low calcium, magnesium-free (LCMF) HBSS. LCMF HBSS was prepared by supplementing CMF HBSS with CaCl2.H2O, 1% BSA, 25 mM HEPES, 0.035% NaHCO3, and penicillin/streptomycin (100 units/ml). The tissue was dissociated into component cells using a modification of the procedure developed by Huang & Miller (1980). Briefly, anterior pituitary tissue was dissected free of connective tissue and the posterior pituitary and sliced into 0.5 mm sections using a Stadie–Riggs tissue slicer. The pituitary slices were minced into roughly 2 mm2 with scissors. The minced tissue from five pituitary glands was combined and washed five times with LCMF HBSS before resuspension into 66 ml LCMF HBSS containing 200 mg collagenase (290 U/mg). The tissue suspension was gently agitated in a rotary shaker (90 r.p.m.) at 37 °C for 90 min. After incubation with collagenase, the partially digested tissue was pelleted by centrifugation (400 g for 4 min). The collagenase containing supernatant was decanted and tissue fragments were resuspended in 60 ml CMF HBSS containing 0.25% pancreatin. Tissue fragments were incubated with pancreatin for 35 min at 37 °C with gentle agitation. At the conclusion of enzyme treatment, undigested tissue was removed by passage of the cell suspension through a nylon mesh filter. Dispersed cells were pelleted by centrifugation (400 g for 4 min) and washed four times with CMF HBSS. Cell yield was assessed using a hemocytometer and viability was determined by dye exclusion. Average yield was 200×106 cells/g initial tissue (n=5), with a viability >90% (mean=95.5%). After the final wash with CMF HBSS, the cell pellet was suspended using M199 containing NaHCO3 (2.2 g/l), l-glutamine (200 mM), insulin (5 μg/ml), gentamycin sulfate (60 μg/ml), penicillin/streptomycin (100 units/ml), fungizone (500 ng/ml), HEPES sodium salt (25 mM), and 10% serum collected from castrated male sheep (M199+10% wether serum (WS)). One milliliter aliquot of the cell suspension (0.5×106 cells/ml) was used to seed the 24-well culture plates. The seeded plates were placed in a water-jacketed incubator and cells were allowed to attach during 36–48 h culture at 37 °C in a humidified atmosphere of 5% CO2 and air.
Experimental procedure
The effect of E2 and PEs on the basal secretion of FSH from attached pituitary cells was assessed by removing conditioned culture media, washing each well with 1 ml M199+10% WS and, finally adding 1 ml M199+10% WS the appropriate concentration of test compound (E2 with PE). The PEs (CM, zearalenone (ZR), GN, DZ, resveratrol (RV), biochanin A (BA), and enterolactone (EL)) were diluted in DMSO to working concentrations (0.001–1.0 μM) using M199+10% WS (final DMSO concentration was 0.2%). Vehicle also contained DMSO. We found no evidence that DMSO at the concentration (0.2%) used in these studies influenced the results. Conditioned media was collected after 48 h incubation with vehicle, E2, or PE. In some experiments vehicle, E2, or PEs were coincubated with an ER antagonist (ICI 182 780). After removal of conditioned media, cellular content of FSH was assessed by adding 1 ml of cold 0.01 M PBS to each well and rapidly freezing plates at −85 °C. The attached cells were lysed by repeated freezing and thawing as described in previous studies (Adams et al. 1979). PBS was collected after the fourth freeze–thaw cycle and used to determine cellular FSH content. The concentration was then combined with basal secretion to determine the total production of FSH.
The effect of E2 and PEs on the sensitivity and magnitude of GnRH-A-induced LH secretion was assessed using a related experimental paradigm. Briefly, after a 48 h attachment period, conditioned media was removed and replaced with 1 ml media containing the desired concentration of E2, PE, and/or ICI. After 12 h incubation with test compounds, wells were flushed twice with 1 ml media, with final addition of 1 ml media containing vehicle or the appropriate concentration of GnRH-A (0.1–1000 pM). Media from treated wells was collected after a 6 h incubation with the GnRH-A.
Measurement of LH and FSH
The concentrations of LH and FSH were determined by RIA following procedures described previously (Adams et al. 1975, 1988). The ovine FSH (NIADDK-oFSH-20) and LH (NIADDK-oLH-26) standard preparations were kindly provided by NIH.
Statistical analysis
The statistical significance of experimental observations was assessed using the mixed model procedure lmer() in R (Bates & Maechler 2009). For studies of basal FSH secretion, fixed effects included treatment, experiment, and treatment–experiment interaction, while plate was included as a random effect. In contrast, GnRH-A-induced LH secretion was analyzed using a model that included the fixed effects of treatment, GnRH, and treatment–GnRH-A interaction; experiment and experiment–GnRH-A, while plate and plate–GnRH-A were included as random effects. The likelihood-ratio-test was used to determine the significance of the fixed effects (P<0.05) and competing models where fitted using maximum likelihood. Contrasts were setup to determine the mean comparisons against the negative control (vehicle) and positive controls (0.05 and 5 nM E2). The Bonferroni correction was used to control the type 2 error rate.
Results
Effect of E2 and PEs on basal gonadotropin parameters
To determine the effects of E2 and PEs on basal secretion of FSH and total FSH production, we incubated ovine pituitary cells with increasing concentrations of E2 and PEs for 48 h. Physiologic (0.05 nM) and pharmacologic (5 nM) concentrations of E2 markedly reduced basal secretion of FSH and significantly reduced total FSH production (Fig. 1). Some, but not all, PEs had a similar effect. The two most potent PEs in this regard were CM and ZR. Neither DZ, RV, BA, nor EL affected basal FSH secretion or total production of FSH in this ovine pituitary cells culture system.
Role of ER on basal FSH parameters
To assess the role of estrogen receptors in expression of the effects of E2 and PEs, we coincubated pituitary cells with E2 or PEs and ICI 182 780 (ICI), a potent estrogen receptor antagonist. The results of these coincubation studies clearly demonstrate that estrogen-dependent suppression of both FSH parameters is mediated by ER (Fig. 2). Indeed, the normal estrogen-induced reduction in FSH secretion was blocked, or markedly attenuated in cells receiving the estrogenic stimulus in combination with ICI. It is important to note that ICI not only did block the response induced by E2 but also prevented the PE-induced decrease in basal FSH secretion (Fig. 2A). Similarly, estrogen-depended decrease in total FSH production was prevented in cells receiving estrogen and ICI concurrently (Fig. 2B).
Effect of E2 and PEs on gonadotrope responsiveness
In another set of studies, we examined the effect of E2 and PEs on the magnitude and sensitivity of GnRH-A-induced LH secretion. As described in detail in the Materials and methods section, attached cells were pretreated with E2, PE, and/or ICI for 12 h and then exposed to vehicle or various concentrations of GnRH-A for 6 h. The magnitude and sensitivity of GnRH-A-induced LH secretion during the 6 h treatment period were determined. The effect of E2, ICI, and selected PEs on gonadotrope responsiveness is illustrated in Fig. 3. These data clearly demonstrate that pretreatment with physiological concentration of E2 significantly enhanced the magnitude and sensitivity of LH secretion induced by GnRH-A. Pretreatment of pituitary cells with selected PEs, including CM, ZR, GN, or DZ, resulted in similar augmentation of gonadotrope responsiveness. The enhanced gonadotrope responsiveness resulting from pretreatment with E2 or PEs was markedly attenuated in cells coincubated with estrogenic stimuli and ICI. Interestingly, pretreatment with ICI alone resulted in a modest, but consistent increase in both maximal GnRH-A dependent LH secretion and gonadotrope sensitivity.
The data presented in Table 1 demonstrate that 12 h pretreatment of pituitary cells with E2 or PEs did not affect subsequent basal secretion of LH. Conversely, the magnitude of LH secretion induced by 1000 pM GnRH-A was significantly increased by prior exposure to physiological concentration of E2 (50 pM). Pretreatment with PEs similarly increased the magnitude of GnRH-A-induced LH secretion. The PEs with the highest potency in this regard were CM and ZR, both of which induced the greatest enhancement of secretory response at a PE concentration of 0.1 μM. The augmented response induced by GN or DZ was somewhat less robust in that 1 μM concentration of the PE was required to elicit significant enhancement of gonadotrope responsiveness.
Effect of estradiol (E2) and increasing concentrations of coumestrol (CM) or zearalenone (ZR) on basal and GnRH-agonist (GnRH-A)-induced LH secretion. After a 48 h period of attachment, pituitary cells were incubated for 12 h with vehicle, E2, or increasing concentrations of CM or ZR. The magnitude of LH secretion induced by 1 nM GnRH-A was assessed during the 6 h period that followed estrogen pretreatment. Values represent the mean±s.e.m. of at least three replicates of the experiment
Pretreatment | Basal LH (ng/well) | 1 nM GnRH-A-induced LH secretion (ng/well) |
---|---|---|
Vehicle | 14.6±0.5* | 45.9±1.9* |
50 pM E2 | 14.6±0.7* | 90.2±5.5† |
Coumestrol (nM) | ||
1 | 11.6±0.6* | 42.4±1.0* |
10 | 13.2±0.8* | 67.9±1.4† |
100 | 12.1±0.5* | 83.1±3.7† |
1000 | 12.1±0.8* | 78.7±4.1† |
Zearalanone (nM) | ||
1 | 11.5±0.7* | 35.8±1.0* |
10 | 13.5±0.3* | 63.4±4.1‡ |
100 | 11.3±0.8* | 77.3±1.2† |
1000 | 12.1±0.4* | 75.6±4.1† |
*,†,‡Values within a column with differing superscripts differed significantly (P<0.05).
In addition to affecting the magnitude of LH secretion induced by high levels of GnRH or GnRH-A stimulation, prior exposure to E2 or PEs may also affect the sensitivity of the response. We quantified gonadotrope sensitivity by determining the concentration of GnRH-A required to establish the half maximal secretory response. The results of these analyses are presented in Table 2. These data demonstrate that the concentration of GnRH-A required to induce half maximal LH secretion from control cells pretreated with vehicle alone was 35.5±2.8 pM. Pretreatment of pituitary cells with physiological concentrations of E2 (50 pM) markedly enhanced gonadotrope sensitivity and, as a consequence, lowered the GnRH-A stimulus required to establish the half maximal response to 1.3±2.8 pM. Pretreatment with selected PEs had a similar effect on gonadotrope sensitivity, with CM and ZR being most effective in this regard. It is again important to note that the augmentation of gonadotrope sensitivity induced by E2 and PEs was partially reversed in cells exposed to estrogen together with ICI during the pretreatment period. The other PEs examined, RV, BA, and EL, did not affect either gonadotrope sensitivity or maximal agonist-induced response, even at maximum concentrations (1 μM).
Effect of estradiol (E2), selected phytoestrogens (PEs), and/or ICI 182 780 (ICI), an estrogen receptor antagonist, on the sensitivity of GnRH agonist (GnRH-A)-induced LH secretion. The concentration of GnRH-A that effected half maximal LH secretion was used as measure of gonadotrope sensitivity. After a 48 h period of attachment pituitary cells were incubated for 12 h with vehicle, E2 (50 pM), coumestrol (CM; 0.1 μM), zearalenone (ZR; 0.1 μM), genistein (GN: 1 μM), or daidzein (DZ; 1 μM DZ) alone or in combination with 1 μM ICI. The magnitude of GnRH-A induced LH secretion was assessed during the 6 h period that followed estrogen pretreatment. Values represent the mean±s.e.m. of at least three replicates of the experiment
Pretreatment | ||
---|---|---|
Estrogen | ICI 182 780 (1 μM) | GnRH-A (pM)a |
Vehicle | Vehicle | 35.5±2.8* |
Vehicle | ICI | 19.1±2.8‡ |
E2 (50 pM) | Vehicle | 1.3±2.8† |
E2 (50 pM) | ICI | 23.7±2.8‡ |
CM (0.1 μM) | Vehicle | 1.2±2.8† |
CM (0.1 μM) | ICI | 20.0±2.8‡ |
ZR (0.1 μM) | Vehicle | 1.3±2.8† |
ZR (0.1 μM) | ICI | 16.7±2.8‡ |
GN (1 μM) | Vehicle | 0.9±2.8† |
GN (1 μM) | ICI | 10.3±2.8§ |
DZ (1 μM) | Vehicle | 1.4±2.8† |
DZ (1 μM) | ICI | 20.0±2.8‡ |
*,†,‡,§Values with differing superscripts differed significantly (P<0.05).
Concentration of GnRH-A required to induce half maximal LH secretion.
Discussion
The results presented here demonstrate that PEs exert estrogen-like effects at pituitary loci. We demonstrated that PEs, like E2 itself, suppressed basal FSH secretion and also reduce total FSH production. Similarly, we demonstrated that PE treatments exhibited estrogen-like activity and increased the secretory response induced by GnRH-A, also in a dose-dependent manner. Furthermore, we combined estrogenic stimuli with the ‘pure’ ER antagonist, ICI, to illustrate that the PE-mediated effects on basal FSH secretion and GnRH-A induced LH secretion are ER-dependent. Interestingly, ICI displayed estrogenic and anti-estrogenic effects on gonadotrope function in the ovine pituitary cell culture system used in our study.
Estrogenic stimuli are known to attenuate basal FSH secretion and residual intracellular FSH and thereby decrease total FSH production. This has been demonstrated in several species using both in vivo and in vitro models (Lindzey et al. 2006, Arreguin-Arevalo et al. 2007). Groups using ovine pituitary cells in culture have clearly demonstrated that treatment with E2 attenuates FSH secretion and intracellular synthesis (Miller et al. 1977, Huang & Miller 1980, Nett et al. 2002). In particular, E2 suppresses these parameters by decreasing FSHβ expression (Miller & Miller 1996, Baratta et al. 2001). Unfortunately, the specific mechanism by which E2 exerts this effect is not clear. However, Baratta et al. (2001) demonstrated that E2 decreased activinβ, a growth factor necessary for normal FSHβ transcription. They concluded that E2 indirectly regulates FSHβ transcription by decreasing activinβ and, ultimately, decreasing synthesis and secretion of FSH. Our results are consistent with this model.
It is well established that estrogenic stimuli increase gonadotrope responsiveness. Elevated E2 concentrations immediately preceding ovulation increase GnRH receptor (GnRHR) expression in gonadotrope cells in sheep (Sakurai & Adams 1991, Turzillo et al. 1998, Clarke 2002). Hypothalamic-derived GnRH binds the GnRHR, thereby activating the necessary second messenger cascades, which ultimately increase intracellular calcium concentrations necessary to facilitate LH secretion (Anderson 1996). Our study demonstrates that certain PEs, particularly CM, ZR, GN, and DZ, induce estrogen-like effects on gonadotrope responsiveness. One explanation is that PEs increase GnRHR mRNA and expression in the pituitary like E2. That this response to E2 or the PEs requires one or both of the ER isoforms is suggested by our observation that the responses are attenuated by coincubation with the ER antagonist ICI.
The ER antagonist is commonly used in vivo and in vitro to determine the role of the ER in physiological processes. Upon binding the ER, ICI prevents the receptor dimerization necessary for gene expression (Dauvois et al. 1993, Osborne et al. 2004). Furthermore, ICI effectively inactivates the ER by blocking the AF1 and AF2 transcription activation domains and ultimately increases ER degradation (Osborne et al. 1995, Wakeling 1995). Collectively, these effects of ICI block the effective action of residual ERs. We observed that ICI blocked the effects of E2 and maximum concentrations of CM, ZR, and GN. Our data illustrate that ICI combined with estrogenic stimuli blocked the suppression of basal FSH secretion and total production of FSH. This suggests that ICI prevents estrogenic stimuli from attenuating activinβ transcription.
Interestingly, ICI exhibited biphasic responses on gonadotrope responsiveness. When estrogenic stimuli were combined with ICI, we recorded a decrease in responsiveness. This observation indicates that E2 and the PEs exert their effect by interacting with one or both of the ER isoforms in gonadotrope cells. In contrast, ICI alone induced a modest, but significant and repeatable, enhancement of gonadotrope responsiveness and suggests that ICI functions as a partial agonist in our in vitro pituitary cell culture system. Although most studies indicate that ICI functions as a pure ER antagonist, recent observations demonstrate that ICI may have partial agonist activity at some tissues. For example, research conducted using rat primary hippocampal neurons also demonstrated that ICI exhibits agonistic activity (Zhao et al. 2006).
It is important to note that our in vitro cell culture system comprises a heterogeneous population of pituitary cells. The gonadotrope cells predominately express ERα and, therefore, are likely to respond directly to estrogenic inputs (Tobin et al. 2001, Clarke 2002). However, many other pituitary cells, including somatotropes and lactotropes, also express ERs and also have the capacity for direct response to estrogens (Tobin et al. 2001, Zárate & Seilicovich 2010) or antiestrogens like ICI. This raises the possibility that paracrine signals from adjacent pituitary cells may impinge on the gonadotropes to modulate various aspects cell of function. In this mixed cell system, the measurable parameters, like gonadotrope responsiveness and basal secretion of LH and FSH, may represent the cumulative effect of direct action of estrogens and antiestrogens on gonadotrope cells and indirect estrogenic responses mediated by estrogen-controlled paracrine factors produced by other pituitary cells.
In conclusion, our study illustrates that PEs decrease basal FSH secretion and total FSH production in a manner that is comparable to E2. This action is blocked when E2 or PE are combined with the ER antagonist, ICI. Our study also shows that PEs increase gonadotrope responsiveness to the GnRH-A in a manner that is similar to E2, and this effect is partially reversed by ICI. Our continuing studies are examining the relative important of specific ER isoforms in the expression of the multifaceted effects of estrogens on gonadotrope function.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research was supported by a HATCH Project grant (RR-4505) from the USDA.
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